This program explores roles of reactive oxygen species (ROS) as specific signaling molecules in B and T lymphocytes through genetic manipulation of NOX/DUOX family NADPH oxidases. These enzymes catalyze NADPH-dependent reduction of molecular oxygen to generate superoxide or hydrogen peroxide. Phagocytes produce large amounts of ROS in response to infectious or inflammatory stimuli through the prototypic NADPH oxidase containing gp91phox (a.k.a., NOX2). Although originally understood as an anti-bacterial mechanism deployed by phagocytes, our research revealed that ROS intentionally generated by several NOX family members play specific signaling roles in B cell receptor (BCR)-stimulated B cells and T cell receptor (TCR)-stimulated T cells. Our studies in lymphocytes are exploring roles of NOX family members in adaptive immune responses to diverse pathogens, as well as in autoimmunity or immunodeficiencies. Several NOX family oxidases have been associated with auto-immune inflammatory disease-like processes, such as inflammatory bowel disease. About 50% of chronic granulomatous disease patients with NOX2 deficiencies suffer from very early onset inflammatory disease (VEOIBD). In 2018, we characterized a novel inactive form of NOX5 identified in a patient with VEOIBD. In collaboration with Kathleen Sullivan (CHOP), we investigated functional effects of a mono-allelic mutation in NOX5 (P323_G325del) identified by total exome sequencing in a patient with severe VEOIBD. The alignment of this NOX5 sequence with other NOX proteins showed that the 3-codon deletion occurs within the fourth transmembrane helix, between heme-binding helices 3 and 5. While predicted to be highly disruptive, the variant exhibits stability comparable to WT NOX5. We showed the mutated protein produces little or no superoxide in three reconstituted cell models (Jurkat, COS-7 and HEK 293 cells) in responses to three different agonists, PMA, calcium/ionomycin or hydrogen peroxide. The mutated protein also causes dose-dependent inhibition of NADPH oxidase activity of co-expressed wild-type NOX5 in two NOX5-reconstituted models (COS-7 and HEK 293 cells), providing further insight on dominant-negative effects of this monoallelic gene variant. Future work will examine NOX5 mRNA and protein expression patterns in healthy and diseased tissue to determine its roles in inflammatory disease. These studies represent the first case in which genetic and functional defects in NOX5 have been linked to human disease.